The Study On The Involvment Of MiR-29b-3p/Glutamate Receotor 4 (GRM4) In Antidepressant Effect Of Ketamine

ObjectivesDepression is a chronic mental illness,which will become the world's second disability by 2020,but the effect of traditional antidepressant treatment is slow onset.Recent studies have indicated that the subanesthestic dosage of ketamine could have a rapid antidepressant effect,however,the relevant mechanism is still unclear.Previous studies found that up-regulation expression of metabolic type glutamate receptor 4(GRM4)in brain region was closely related to the onset and development of depression disorder.In addition,the related studies and our previous results found that the up-regulation expression of miR-29a/b/c-3p was induced by the subanesthestic dosage of ketamine and the bioinformatics method predicted that GRM4 may be target gene of miR-29a/b/c-3p.Therefore,in order to provide a new approach and potential therapeutic target gene for depression treatment in future,we explored whether the up-regulation expression of miR-29a/b/c-3p of related brain regions was induced by ketamine,and further resulted in the down-regulation expression of glutamate receotor 4(GRM4),which was related to the antidepressant effect of ketamine.Methods 1.qRT-PCR was used to detect expression of miR-29a/b/c-3p of the prefrontal cortex,hypothalamus and hippocampus in the normal SD rats treated by subanesthestic dosage of ketamine.2.Dual-Luciferase Reporter Assay was used to verify whether the related miRNAs directly target GRM4.3.Unpredictable chronic mild stress method was used to establish the rat model of depression.After treatment of subanesthestic dosage of ketamine,related behavioral assessment was performed.4.qRT-PCR was used to detect expression of differential miR-29a/b/c-3p of the special brain region in depression-like rats and depression-like rats treated by subanesthestic dosage of ketamine.5.qRT-PCR and Western blotting were used to respectively detect expression of GRM4(m RNA and protein)of the special brain region in depression-like rats and depression-like rats treated by subanesthestic dosage of ketamine.Results1.After the normal SD rats were treated by subanesthestic dosage of ketamine:(1)The expression of miR-29b-3p was increased in the prefrontal cortex of normal rats(p < 0.05),but had no statistical difference in the hypothalamus and hippocampus of normal rats(p > 0.05).(2)The expression of miR-29a/c-3p had no statistical difference in the prefrontal cortex,hypothalamus and hippocampus of normal rats(p > 0.05).2.The result of dual luciferase reporter confirmed miR-29b-3p could directly target 3 'UTR region of GRM4,after the seed sequence mutation,the target function disappeared.3.Chronic mild unpredictable stress resulted in the decline of sugar water consumption,the decrease of total distance and the increase of immobility time of normal SD rats,but the sugar water consumption and the total distance were increased and the immobility time was shortened by subanesthestic dosage of ketamine in depression-like rats(p < 0.05).4.The expressin of miR-29b-3p in prefrontal cortex was reduced(p < 0.01),but had no statistical difference in the hypothalamus and hippocampus in depression-like rats(p > 0.05).After depression-like rats were treated by subanesthestic dosage of ketamine,the expression of miR-29b-3p in prefrontal cortex was obviously increased(p < 0.01).5.The expression of GRM4(m RNA and protein)in prefrontal cortex was increased in the depression-like rats(p < 0.01).After depression-like rats were treated by subanesthestic dosage of ketamine,the expressin of GRM4(m RNA and protein)in prefrontal cortex was reduced(p < 0.01).Conclusions 1.Ketamine induces the expression of miR-29b-3p in the prefrontal cortex of rat.2.GRM4 is the target gene of miR-29b-3p.3.Mi R-29b-3p/GRM4 in the prefrontal cortex is involved in antidepressant effect of ketamine.